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A Cayota

Publications and source records attributed to A Cayota.

18 recordsLinked to original sources
PubMed2005

Protein 3-nitrotyrosine formation during Trypanosoma cruzi infection in mice.

Nitric oxide (.NO) is a diffusible messenger implicated in Trypanosoma cruzi resistance. Excess production of .NO and oxidants leads to the generation of nitrogen dioxide (.NO2), a strong nitrating agent. Tyrosine nitration is a post-translational modification resulting from the addition of a nitro (-NO2) group to the ortho-position of tyrosine residues. Detection of protein 3-nitrotyrosine is regarded as a marker of nitro-oxidative stress and is observed in inflammatory processes. The formation and role of nitrating species in the control and myocardiopathy of T. cruzi infection remain to be studied. We investigated the levels of .NO and protein 3-nitrotyrosine in the plasma of C3H and BALB/c mice and pharmacologically modulated their production during the acute phase of T. cruzi infection. We also looked for protein 3-nitrotyrosine in the hearts of infected animals. Our results demonstrated that C3H animals produced higher amounts of .NO than BALB/c mice, but their generation of peroxynitrite was not proportionally enhanced and they had higher parasitemias. While N G-nitro-arginine methyl ester treatment abolished .NO production and drastically augmented the parasitism, mercaptoethylguanidine and guanido-ethyl disulfide, at doses that moderately reduced the .NO and 3-nitrotyrosine levels, paradoxically diminished the parasitemia in both strains. Nitrated proteins were also demonstrated in myocardial cells of infected mice. These data suggest that the control of T. cruzi infection depends not only on the capacity to produce .NO, but also on its metabolic fate, including the generation of nitrating species that may constitute an important element in parasite resistance and collateral myocardial damage.

Acute Disease↗
PubMed2004

Post-transcriptional regulation of inducible nitric oxide synthase in chronic lymphocytic leukemia B cells in pro- and antiapoptotic culture conditions.

Functional inducible NOS (iNOS) may be involved in the prolonged lifespan of chronic lymphocytic leukemia cells (B-CLL), although the exact mechanisms implicated remain elusive as yet. In this work, we have examined iNOS expression in normal B lymphocytes and B-CLL cells in pro- and antiapoptotic conditions. Our results demonstrate: (1) The existence of a new splice variant characterized by a complete deletion of exon 14 (iNOS 13-16(14del)), which was preferentially detected in normal B lymphocytes and may represent an isoform that could play a role in the regulation of enzyme activity. (2) The existence of another alternatively spliced iNOS mRNA transcript involving a partial deletion of the flavodoxin region (iNOS 13-16(neg)) was correlated to a decreased B-CLL cell viability. The 9-beta-D-arabinofuranosyl-2-fluoradenine or fludarabine (F-ara) treatment induced iNOS 13-16(neg) transcript variants, whereas IL-4 enhanced both the transcription of variants, including these exons (iNOS 13-16(pos)), and the expression of a 122 kDa iNOS protein. These results suggest that in B-CLL, a regulation process involving nitric oxide (.- NO) levels could occur by a post-transcriptional mechanism mediated by soluble factors. Our results also provide an insight into a new complementary proapoptotic action of F-ara in B-CLL by the induction of particular iNOS splice variants, leading to the activation of a caspase-3-dependent apoptotic pathway.

Aged↗
PubMed2001

Unraveling peroxynitrite formation in biological systems.

Peroxynitrite promotes oxidative damage and is implicated in the pathophysiology of various diseases that involve accelerated rates of nitric oxide and superoxide formation. The unambiguous detection of peroxynitrite in biological systems is, however, difficult due to the combination of a short biological half-life, limited diffusion, multiple target molecule reactions, and participation of alternative oxidation/nitration pathways. In this review, we provide the conceptual framework and a comprehensive analysis of the current experimental strategies that can serve to unequivocally define the existence and quantitation of peroxynitrite in biological systems of different levels of organization and complexity.

Animals↗
PubMed2000

Production and functional characterization of two mouse/human chimeric antibodies with specificity for the tumor-associated Tn-antigen.

In this work, we have constructed two functional mouse/human chimeric antibodies (IgMkappa and IgG1kappa isotypes) by inserting genomic DNA fragments encoding VH and Vkappa variable regions of the murine monoclonal antibody IgMK-83D4 into mammalian expression vectors containing human mu, gamma1, and kappa constant exons, and by transfecting them into the nonsecreting mouse myeloma X-63 cell line. In previous works, we have demonstrated that 83D4 murine mAb reacts with Tn determinant (GalNAcalpha-O-Ser/Thr) expressed in 90% of breast, ovary, and colon carcinomas. Both expressed chimeric antibodies were purified from the transfected cell line supernatant by affinity chromatography, and their reactivities against Tn antigen were confirmed by ELISA on asialo ovine submaxilar mucin and immunofluorescence studies on MCF-7 breast carcinoma cell line. We have demonstrated by gel filtration chromatography, that the principal secreted forms were monomers for IgG1kappa and pentamers for IgMkappa. The binding affinities of these chimeric antibodies against synthetic Tn glycopeptides, were evaluated by surface plasmon resonance showing an affinity constant similar to that of 83D4 native antibody for IgMkappa and a lower affinity constant for IgG1kappa chimeric antibody. On the other hand, the replacement of mouse C regions with human C regions confers both chimeric antibodies the ability to activate human complement. These mouse/human chimeric antibodies should be much less immunogenic and could play an important role in the lysis of tumor cell expressing Tn-antigen. Therefore, these anti-Tn chimeric antibodies could be considered as potential tools for human in vivo studies.

Animals↗
PubMed1998

Nitric oxide and peroxynitrite-dependent aconitase inactivation and iron-regulatory protein-1 activation in mammalian fibroblasts.

The reaction of reactive oxygen and nitrogen species with the [4Fe-4S]2+ cluster of mitochondrial (m-) and cytosolic (c-) aconitases leads to loss of catalytic activity and, in the case of the c-aconitase, triggers total cluster disruption to yield the iron-regulatory protein-1 (IRP-1). Herein we have studied the relative contribution and interplay of reactive oxygen species (O and H2O2), nitric oxide (*NO), and peroxynitrite in the modulation of m- and c-aconitase and IRP-1 activities in V79-M8 mammalian fibroblasts, identifying key variables that control the various reactivities at the cellular level. Extracellular production of H2O2 led to inactivation of both m- and c-aconitase and IRP-1 activation, while extracellular had no effect. However, increased intracellular production of caused a loss in m- and c-aconitase activity and IRP-1 activation. Nitric oxide released from NOC-12 had a more complex effect on aconitase and IRP-1 activities. Mitochondrial aconitase was more sensitive than c-aconitase to *NO-mediated inactivation and minimal activation of IRP-1 was observed during a 30-min exposure to the *NO donor. The action of *NO was down- or upregulated by the presence of extra- or intracelular, respectively. Extracellular decreased the *NO-mediated inactivation of aconitases, due to the preferential extracellular decomposition and the lower diffusivity of peroxynitrite compared to *NO. On the other hand, *NO exposure concomitant with enhanced intracellular fluxes lead to intracellular peroxynitrite formation as evidenced by Western blot analysis of nitrated proteins, which increased the effects observed with *NO alone. Peroxynitrite-mediated aconitase inactivation, IRP-1 activation, and cellular protein nitration were more pronounced in cells with low GSH content such as V79-M8 glutathione-depleted cells as well as in pGSOD4 cells which contain 32% of the GSH of the parental strain. Mechanistically, our results imply that the differential actions of the studied reactive species toward cellular aconitases depend on at least three critical factors: (i) their reaction rates with aconitases, (ii) the cellular compartment where they are formed, and (iii) the intracellular status of glutathione.

Aconitate Hydratase↗
PubMed1997

Expression of the IL-2 receptor gamma subunit in resting human CD4 T lymphocytes: mRNA is constitutively transcribed and the protein stored as an intracellular component.

IL-2 receptor (IL-2R) is composed of three subunits named IL-2R alpha, IL-2R gamma. Here, we study the expression of the IL-2R gamma in highly purified, resting peripheral human CD4 T lymphocytes. We show by FACS analysis that the IL-2R gamma subunit is not detectable at the cell surface of peripheral CD4 T lymphocytes. This result has been verified after acid treatment of the cell surface and analysis with three specific anti-IL-2R gamma mAb. Using RT-PCR and intracellular FACS analysis, we demonstrate that IL-2R gamma is constitutively expressed at the mRNA level and the protein is stored as an intracellular component in resting CD4 T lymphocytes. IL-2R alpha and beta subunits are not detectable by these methods. In addition, we show that CD4 T cell remain insensitive to a variety of cytokines that share IL-2R gamma as a common subunit of their receptors (e.g. IL-2, IL-4, IL-7 and IL-15). The kinetics of cell surface expression of IL-2R gamma have been studied after activation of CD4 T lymphocytes and compared with induction of IL-2R alpha and IL-2R beta. Maximum expression of IL-2R gamma is observed after 2 days of stimulation, and remains constant and comparable to IL-2R beta up to day 5. We conclude from these studies that IL-2R gamma is translocated to the membrane only after T cell activation and induction of the IL-2R alpha and IL-2R beta genes. We hypothesize that in CD4 T cells a large intracellular pool of IL-2R gamma is present but that its cell surface translocation depends on the expression of alpha and/or beta chains specific for IL-2, IL-4, IL-7, IL-9 or IL-15.

CD4-Positive T-Lymphocytes↗
PubMed1996

In vitro antioxidant treatment recovers proliferative responses of anergic CD4+ lymphocytes from human immunodeficiency virus-infected individuals.

Oxidative stress has been proposed to be involved in the immunologic defeat observed in effector calls of the immune system as well as in lymphocyte cell death and viral replication in human immunodeficiency virus (HIV)-infected patients. Because thiol-containing antioxidants such as N-acetyl-L-cysteine have been shown to have beneficial effects on CD4+ lymphocyte survival and to inhibit programmed cell death and HIV-1 replication, they may play a role in therapeutic strategies of this disease. In this work we have studied the cellular thiol levels and the affect of in vitro antioxidant treatment of purified CD4+ lymphocytes from HIV-infected patients, and correlated these parameters to proliferative responses and programmed cell death. We show that CD4+ lymphocytes from HIV-infected patients display impaired proliferative responses and a significant decrease in cellular thiol levels, indicating a disturbed redox status. Interestingly, antioxidant treatment succeeded to restore defective proliferative responses to CD3-mediated activation in 8 of 11 patients (high antioxidant responders). In contrast to high responders, patients failing to respond to antioxidant treatment (low antioxidant responders), were characterized by an abnormal ratio of apoptotic cells, which was not affected by N-acetyl-L-cysteine and/or 2-beta-mercaptoethanol preincubation. These results demonstrate for the first time that antioxidant treatment is able to revert the impaired proliferative activity of CD4 cells from HIV-infected patients and could help designing therapeutic strategies with antioxidant drugs. However, this action is not observed in cells undergoing programmed cell death.

Acetylcysteine↗
PubMed1996

CD4+ lymphocytes from HIV-infected patients display impaired CD45-associated tyrosine phosphatase activity which is enhanced by anti-oxidants.

It has been proposed that signal transduction defects may, at least partially, account for the functional impairment of CD4+ lymphocytes during HIV-1 infection. Recently, we have demonstrated that unresponsive CD4+ lymphocytes from these patients had reduced protein tyrosine phosphorylation after CD3 engagement, and that this defect was associated with constitutively altered levels of p56lck and p59fyn kinases. Since CD45 is essential for T cell receptor (TCR) and CD2-mediated activation of protein tyrosine kinases, we study here CD45-associated tyrosine phosphatase activity in resting and activated CD4 T cells from HIV-infected patients. We found a significant decrease in the basal and post-activation phosphatase activity of CD45 which correlated well with impairment of proliferative responses. In addition, decreased levels of cellular thiols observed in resting CD4+ lymphocytes from these patients suggested a disturbed redox status. Although expression levels of CD45 were decreased in most patients, a significant recovery of phosphatase activity and proliferative responses was observed in most patients by preincubating cells with N-acetyl-L-cysteine and beta2-mercaptoethanol. In some patients, anti-oxidant treatment failed to significantly enhance phosphatase activity and proliferative responses. The low responses of purified CD4+ lymphocytes from these patients were associated with a high ratio of apoptotic cell death which did not appear to be influenced by anti-oxidant treatment.

Apoptosis↗
PubMed1995

Flow cytometric analysis of protein-tyrosine phosphorylation in peripheral T cell subsets. Application to healthy and HIV-seropositive subjects.

This paper describes an improved method for detecting tyrosine phosphorylation levels in T cell subsets by flow cytometry early after CD3 crosslinking stimulation. It consists in introducing gentle paraformaldehyde fixation between CD3 crosslinking in cold conditions and the shift to 37 degrees C, which activates downstream signalling machinery. We used the combined properties of monoclonal antibodies for stimulating cells and for detecting surface markers to analyze protein-tyrosine phosphorylation levels in T cells subsets following stimulations which mimic physiological activation. Overall data obtained in healthy subjects, using two- or three-color immunofluorescence, indicated that: (1) most CD3 positive cells phosphorylate tyrosine substrates following CD3 crosslinking stimulation and (2) helper cells phosphorylate tyrosine to a slightly better extent than cytotoxic cells after CD3 crosslinking. Nevertheless, the two subsets follow similar kinetic patterns and tend to retain a homogeneous profile. Processing of samples from HIV-seropositive patients showed heterogeneous phosphorylation levels in both subsets, when compared to normal donors. This assay should, in the future, lead to easy and rapid exploration of the signal transduction pathway in different subsets of T cells under normal and pathological conditions.

CD3 Complex↗
PubMed1994

Defective protein tyrosine phosphorylation and altered levels of p59fyn and p56lck in CD4 T cells from HIV-1 infected patients.

Early in HIV infection, CD4+ lymphocytes exhibit the properties of an anergic state characterized by unresponsiveness to mitogens or to TCR stimulation and by defective IL-2 production. As tyrosine phosphorylation is the earliest of the biochemical events initiated by stimulation of CD3-TCR, we studied protein tyrosine phosphorylation in purified CD4+ lymphocytes from 25 asymptomatic seropositive patients (CD4 T cells > 350/mm3) previously stimulated in vitro by immobilized anti-CD3 mAb or by co-immobilized anti-CD3 and anti-CD28 mAbs. Purified CD4+ lymphocytes from HIV-infected patients exhibited defective early protein tyrosine phosphorylation in response to CD3 activation when compared with normal subjects. This defect was observed mainly in patients in whom proliferative responses to immobilized anti-CD3 ranged from 2 to 50% of control values obtained in healthy donors, and was frequently associated with increased cellular levels of p59fyn and decreased cellular levels of p56lck. Interestingly, these defects appeared to correlate with the degree of impairment in thymidine incorporation. Since CD28 mAbs have been reported to enhance proliferative responses to the CD3-TCR pathway in cloned murine or human anergic models and to induce tyrosine phosphorylation in human T cells, we studied the role of CD28 mAb as a co-signal. Although anti-CD28 co-stimulation augmented the proliferative responses in both controls and HIV-infected patients, it failed to correct the tyrosine phosphorylation pattern in the latter. Our results suggest a relationship between defective early protein tyrosine phosphorylation and impairment of proliferative responses in CD4 T cells from HIV-infected patients, and evidence is provided that associated altered cellular levels of the fyn and lck tyrosine kinases might play an important role in the anergic response observed early during HIV infection.

CD4-Positive T-Lymphocytes↗
PubMed1993

In vitro non-productive infection of purified natural killer cells by the BRU isolate of the human immunodeficiency virus type 1.

Highly purified natural killer (NK) cell lines and clones, displaying the typical phenotype, morphology and function and obtained from healthy blood donors, were infected in vitro with the BRU isolate of human immunodeficiency virus type 1 (HIV-1). There was no significant increase in reverse transcriptase activity and levels of p24 antigen in the supernatants, but positive staining was observed using an immunogold technique with polyclonal anti-HIV-1 antibodies. When infected NK cells were co-cultivated with autologous non-infected CD4+ mitogen-activated cells, significant levels of reverse transcriptase activity and p24 antigen in supernatants were detected. Giant syncytial cells and a high number of mature virion particles were also evident. When NK cell lines or clones from HIV-1-infected patients were studied, neither the presence of p24 antigen nor reverse transcriptase activity was detected in the supernatants after stimulation with mitogens, cytokines or co-culture with allogeneic CD4+ mitogen-activated cells. PCR studies did not detect HIV-1 genes in freshly purified NK cells, cell lines or clones from infected patients. Taken together these results suggest that (i) normal NK cells can be infected in vitro by the HIV-1 BRU isolate in a non-productive fashion, (ii) PCR with NK cell DNA of HIV-1-infected patients indicates that in vivo few of these cells, if any, are infected by HIV-1 and (iii) the mechanisms responsible for the impairment of NK cell function during HIV-1 infection remain to be determined and are probably not related to a direct cytopathic effect of the virus.

Cell Line↗
PubMed1993

Differential requirements for HIV-1 replication in naive and memory CD4 T cells from asymptomatic HIV-1 seropositive carriers and AIDS patients.

One of the major routes for modulating HIV-1 expression by infected T cells is through the control of transcription initiation from the HIV-1 long terminal repeat (LTR), which is regulated either by its own viral gene products or by several cellular DNA-binding proteins induced during T cell activation. Previous work reported preferential HIV-1 infection and replication of memory CD4 T cells from infected individuals, which was explained either by a higher viral burden of this subset or by differences between naive and memory cells in the activation of the general transcription machinery involved in HIV-1 replication. In this work, we have studied HIV-1 replication by highly purified naive and memory CD4 T cells from asymptomatic seropositive carriers (ASC) and AIDS patients following different activation signals. Our results demonstrate that viral replication in memory cells from ASC was observed after mitogenic (phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA)) recombinant tumour necrosis factor-alpha (rTNF-alpha) and CD3-mediated activation. In contrast, in naive subsets, early viral replication was almost exclusively observed upon CD3-mediated activation. AIDS patients are characterized by similar levels of viral replication in both subsets after PHA and soluble or immobilized anti-CD3 MoAb activation. However, naive subsets from AIDS patients still displayed differential requirements since they failed to replicate HIV-1 after treatment with PMA and rTNF-alpha. Taken together, these results provide evidence that HIV-1 replication in CD4+ T cells from infected individuals is a function of the differentiation stage of the cells, the disease stage of the patient and the activation signal employed.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Impaired proliferative capacity and abnormal cytokine profile of naive and memory CD4 T cells from HIV-seropositive patients.

Purified naive and memory CD4 T cells from healthy donors, HIV+ asymptomatic carriers and AIDS patients were examined for their proliferative activity and their pattern of cytokine secretion (IL-4, IL-6, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha)) upon stimulation with phytohaemagglutinin (PHA), phorbol myristate acetate (PMA) and cross-linked anti-CD3 MoAb, in the presence of recombinant IL-2 (rIL-2). We found a decrease in the proliferative capacity of naive CD4 T cells following stimulation with PHA and PMA, and a sharp decline in this response upon cross-linked anti-CD3 stimulation in both subsets, although it predominated in the naive subpopulation. In AIDS patients, less pronounced impairment of thymidine uptake by the naive subset was found upon PHA and cross-linked anti-CD3 MoAb stimulation. In addition, an altered secretion pattern of the different cytokines was observed, consisting of abnormal secretion of IL-6 by both naive and memory cells, an abnormal pattern of IFN-gamma secretion and frequent loss of detectable IL-4 production by HIV patients. These abnormalities were even more pronounced in AIDS patients than in the asymptomatic carriers. Overall, our results extend previous reports indicating functional impairment of memory CD4 subsets in HIV+ subjects by showing that this impairment involves naive CD4 T cells.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Natural killer (NK) cell activity during HIV infection: a decrease in NK activity is observed at the clonal level and is not restored after in vitro long-term culture of NK cells.

NK cell activity is impaired in HIV-infected patients. The mechanisms behind the altered NK functions are not clear, and conflicting data concerning NK and antibody-dependent cellular cytotoxicity (ADCC) activity have been reported. In order to investigate whether this impairment is also observed at the clonal level and whether it is related to a defect at the target cell binding and/or the post-binding level, we evaluated highly purified NK cell lines and cloned NK cells obtained from 22 HIV-infected patients at different stages of disease and compared them with normal controls for their ability to: (i) kill K-562 and U-937 cell lines using a 51Cr release assay; (ii) bind and kill K-562 and U-937 cells at the single cell binding level; (iii) release NK cytotoxic factor (NKCF), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma); (iv) kill anti-IgM preincubated Daudi cell line (ADCC activity). This study with cloned NK cells or NK cell lines from HIV-infected individuals showed: (i) a decrease in their lytic capability against target cell lines; (ii) a low ability to form conjugates with K-562 and U-937 cell lines with respect to controls; (iii) a decreased ability to kill bound target cells; (iv) low levels of released NKCF, TNF-alpha and IFN-gamma after incubation with U-937 cells. Taken together, these findings suggest that the impaired NK cell function during HIV infection is also observed at the clonal level and is related to defects both at the target and post-binding levels. However, the precise mechanisms remain to be determined. The inability to restore normal NK activity after long-term culture in the presence of high levels of recombinant IL-2 is in agreement with the hypothesis of a 'general anergic process' during HIV infection.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Lactate dehydrogenase activity and its isoenzymes in concentric layers of adult bovine and calf lenses.

The activity of lactate dehydrogenase (LDH) and its isoenzyme pattern were studied in four concentric layers of adult bovine and calf lenses. In both groups the specific activity of the total LDH diminished progressively toward the internal nuclear layer; the decrease was greater in the adult lenses. The enzyme activities in the cortical layers of the calf lens were lower than in the adult lens, but in the inner nuclear layers, the opposite was found. All of the 5 LDH isoenzymes were found in each layer. In both groups of animals the LDH1 isoenzyme prevailed, followed by LDH2. No differences were found in the percentage of each isoenzyme in the different lens layers. The differences in the activitie(s) of LDH found may be due to post-translational or post-synthetic modifications which may occur during the aging process.

Aging↗
PubMed1983

The state of sulphydryl groups in lenses of insulin-induced hypoglycemic rats.

The level of non-protein sulphydryl compounds (NP-SH) and protein sulphydryls (P-SH) was investigated in lenses of rats with an acute and extended insulin induced hypoglycemia. No difference was found in the level of NP-SH between the controls and the rats with an acute hypoglycemia induced by cristalline insulin. After 10 hours of hypoglycemia induced by NPH insulin, the decrease of NP-SH was very significant (P less than 0.001). The level of P-SH in lenses of the animals treated with cristalline insulin was found to be lower than in the controls (P less than 0.01), the difference being more significant in the animals treated with NPH insulin (P less than 0.001). The possible reason for these findings is discussed.

Animals↗